Sugar – starch – and carbohydrates – Processes – Carbohydrate manufacture and refining
Reexamination Certificate
2001-12-05
2003-10-28
Kifle, Bruck (Department: 1624)
Sugar, starch, and carbohydrates
Processes
Carbohydrate manufacture and refining
C127S046200, C562S433000, C562S554000
Reexamination Certificate
active
06638360
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the separation of racemic mixtures of optical isomers and other similar molecules into their separate isomeric components. More specifically, the present invention relates to methods for the separation of optical isomers employing differential partitioning of the molecules between high and low density water microdomains.
BACKGROUND OF THE INVENTION
Most biomolecules have at least one asymmetric carbon atom and, therefore, exist as optical isomers, known as enantiomers, which are mirror images of each other. Many synthetic drugs also contain asymmetric carbon atoms. However, since their synthesis is abiotic, the synthetic product is a racemic mixture consisting of equal concentrations of enantiomers. Since proteins and other biologically active molecules have chiral centers at which the molecules act, enantiomers generally have different biological actions, with one enantiomer being more effective than the other. In some cases the enantiomers appear to be antagonistic.
One solution to this problem is to carry out syntheses which result in a single enantiomer, such as those performed by Sepracor Inc. (Marlborough, Mass.). However, such methods are often tedious and expensive. The other solution is to resolve the racemic mixture into separate isomeric compounds. This is difficult to achieve on a preparative scale, with methods presently used for such separations generally relying on specific interactions of the enantiomers on a chiral column. While many drugs are now resolved into their separate isomers during synthesis, there remains a need for a simple and inexpensive method for the resolution of any racemic mixture.
SUMMARY OF THE INVENTION
The present invention provides methods for the separation of enantiomers in a racemic mixture by differentially partitioning the enantiomers into regions of low density water and high density water abutting a porous surface. In preferred embodiments, the porous surface comprises a matrix of small-pored beads. Preferably, the beads comprise pores less than 5 nm in diameter, more preferably less than 3 nm in diameter and most preferably between 1 and 3 nm in diameter.
Matrices of small-pored beads, such as polyamide gels and ion exchange resins, contain separated microdomains of high and low density water. Solutes can be classified into chaotropes or kosmotropes according to their partitioning between these microdomains. Chaotropes partition selectively into low density water and induce high density water, while kosmotropes partition into high density water and induce low density water. In order to separate chaotropes from kosmotropes, the microdomains of high and low density water must be stabilized by balancing chaotropes against kosmotropes and preventing creation of osmotic pressure gradients between the microdomains. When the microdomains are stabilized, the matrix, or gel, is also at its maximum volume. At the particular solution composition at which this balance is achieved, chaotropes, such as D-glucose and L-amino acids, are retained on a polyamide gel and can then be eluted using a solution of a strong chaotrope which breaks down low density water.
More specifically solutes which can break down low density water in a polyamide gel, and can therefore be used to elute chaotropes, include the chaotropes K
+
, Rb
+
, Cs
+
, HCO
3
−
, H
2
PO
4
−
, NO
3
−
, HSO
4
−
, and tetramethyl ammonium ion. Solutes which stabilize low density water in a polyamide gel, and which can therefore be used to elute kosmotropes, include Mg
2+
, Ca
2+
, H
+
, Li
+
, Na
+
, SO
4
2−
, HPO
4
2−
, F
−
, OH
−
and hydrophobic solutes such as ethanol, propanol, benzyl alcohol and butanol.
Small-pored cation and anion ion exchange resins contain microdomains of both high and low density water. The stability of these microdomains in cation exchange resins increases with change of counter ion from Na
+
, Li
+
or H
+
to K
+
, Rb
+
or Cs
+
and especially to Ca
2+
or Mg
2+
. The stability of the microdomains in anion exchange resins increases with change of counter ion from OH
−
or F
−
to Cl
−
, Br
−
or I
−
and especially to SO
4
2−
. Chaotropes, such as L-amino acids and D-glucose, can be eluted from ion exchange resins with aqueous solutions of kosmotropes, such as Mg
2+
, Ca
2+
, H
+
, Li
+
, Na
+
, SO
4
2−
, HPO
4
2−
, F
−
and OH
−
and hydrophobic solutes such as ethanol, propanol, benzyl alcohol and butanol. Kosmotropes, such as D-amino acids and L-glucose, can be eluted from ion exchange resins with solutions of chaotropes, such as K
+
, Rb
+
, Cs
+
, HCO
3
−
, H
2
PO
4
−
, NO
3
−
HSO
4
−
, and tetramethyl ammonium ion. In addition, all solutes, both kosmotropic and chaotropic, can be eluted from ion exchange resins by neutralizing the resins.
In one aspect, the present invention thus provides a method for separating a mixture of a kosmotropic enantiomer and a chaotropic enantiomer comprising: (a) providing a matrix of small-pored beads; (b) stabilizing microdomains of high density water and low density water within the matrix; (c) contacting the stabilized matrix with the mixture of enantiomers; (d) contacting the matrix with a solution that breaks down low density water in the matrix, wherein the chaotropic enantiomer is eluted from the matrix; and (e) contacting the matrix with a solution that stabilizes low density water in the matrix, whereby the kosmotropic enantiomer is eluted from the matrix.
The above-mentioned and additional features of the present invention and the manner of obtaining them will become apparent, and the invention will be best understood by reference to the following more detailed description, read in conjunction with the accompanying drawings.
REFERENCES:
patent: 1007665 (1965-10-01), None
Zolotarev et al, “Ligand Exchange Chromatography for Analysis and Preparative Separation of Tritium-Labelled Amino Acids” J of Radioanalytical and Nuclear Chem., Articles. vol. 121(2), PP. 469-478 (1988).*
Wiggins, Phillipa, “High and Low Density Water in Gels” Prog. Polym. Sci. vol. 20, pp. 1121-1163. (1995).*
Wiggins, Philippa M., “High and Low Density Intracellular Water”,Cellular and Molecular Biology, vol. 47, No. 5, pp. 735--744 (2001).
Biostore New Zealand Limited
Kifle Bruck
Sleath Janet
Speckman Ann W.
Tucker Zachary C.
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